摘要：Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.

摘要：The cis-acting regulatory elements, e.g., promoters and ribosome binding sites （RBSs） with various desired properties, are building blocks widely used in synthetic biology for fine tuning gene expression. In the last decade, acquisition of a controllable regulatory element from a random library has been established and applied to control the protein expression and metabolic flux in different chassis cells. However, more rational strategies are still urgently needed to improve the efficiency and reduce the laborious screening and multifaceted characterizations. Building precise computational models that can predict the activity of regulatory elements and quantitatively design elements with desired strength have been demonstrated tremendous potentiality. Here, recent progress on construction of cis- acting regulatory element library and the quantitative predicting models for design of such elements are reviewed and discussed in detail.

摘要：The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolitesor therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitativecharacterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used *** leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expressionof firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporterprotein and mRNA levels. In addition, we investigated the effects of terminator–promoter combinations on gene expressionand found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest andweakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterizedpromoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expressionstrengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0–362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequencesin N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.